Various techniques are used for the detection and analysis of hydrolyzing enzymes such as amylase. One useful technique is a wet chemistry assay technique involving a starch having a detectable material such as a colored or dye material attached thereto. Such an assay is described in U.S. Pat. No. 3,694,318. In that assay, a dyed starch (a starch having a preformed dye or dye former chemically attached) is dissolved or suspended in a liquid medium. The sample to be tested is then added to the liquid medium, whereupon the amylase present in the test sample hydrolyses the starch, causing release of the dye. The non-hydrolyzed starch is then removed separated and the amount of amylase present as a function of the amount of dye released is determined.
This dyed starch assay has been incorporated into dry test elements, as disclosed in U.S. Pat. No. 4,144,306. The element of that patent comprises: (i) a reagent layer containing a non-diffusible starch having attached thereto a detectable material such as a dye and (ii) a registration layer adapted to receive the detectable material. The sample of liquid to be tested is applied to the reagent layer, whereupon amylase in the sample hydrolyzes the starch, causing it to break down to low molecular weight polysaccharide units with the dye attached thereto. By virtue of their molecular weight, which is lower than that of the non-diffusible starch, the dye-containing low molecular weight polysaccharide units diffuse to the registration layer, where the amount of color formation and thus, the mount of amylase in the sample can be determined.
Dry test elements for the assay of hydrolyzing enzymes such as amylase, like the element described above, have been very successful because of their ease of use and because of fast and reliable results. However, when the test elements have been kept for a period of a week or more at ambient conditions, they have a tendency to overpredict the enzyme concentration as compared to freshly prepared elements or elements that have been stored under refrigeration. The present invention substantially alleviates the problem of overprediction of enzyme concentration in elements that have been stored at ambient conditions.